HEK293T cells were harvested 48 hours after transfection in Phosphate-Buffer Saline (PBS; Severn Biotech LTD) complemented with phosphatase inhibitors (PhoSTOP; Roche) and proteinase inhibitors (COMPLETE; Roche). LCLs were harvested by collecting cells in 15 mL tube (Falcon), centrifuged to form a pellet and resuspended in PBS complemented with phosphatase inhibitors (PhoSTOP; Roche) and proteinase inhibitors (COMPLETE; Roche). Cells were then lysed and processed as previously described (Scotter et al., 2014 (link)). Membrane imaging was conducted using goat anti-rabbit and anti-mouse IgG (H+L) DyLight 680 Conjugate (cat. no. 35568 and 35521, Thermo Life Sciences) and an LI-COR Odyssey or using horseradish peroxidase secondary antibodies for mice (Millipore, 12-349) or rabbits (Millipore, 12-348) and developed through an Enhanced Chemiluminescence System using Medical Film Processor SRX-101A (Konica Minolta). Western blot quantification was performed using the image analysis software, ImageJ (http://imagej.nih.gov/ij/(Schindelin et al., 2012 (link))).
de Majo M., Topp S.D., Smith B.N., Nishimura A.L., Chen H.J., Gkazi A.S., Miller J., Wong C.H., Vance C., Baas F., ten Asbroek A.L., Kenna K.P., Ticozzi N., Redondo A.G., Esteban-Pérez J., Tiloca C., Verde F., Duga S., Morrison K.E., Shaw P.J., Kirby J., Turner M.R., Talbot K., Hardiman O., Glass J.D., de Belleroche J., Gellera C., Ratti A., Al-Chalabi A., Brown R.H., Silani V., Landers J.E, & Shaw C.E. (2018). ALS-associated missense and nonsense TBK1 mutations can both cause loss of kinase function. Neurobiology of Aging, 71, 266.e1-266.e10.
Other organizations :
UK Dementia Research Institute, King's College London, Leiden University Medical Center, University of Amsterdam, University of Massachusetts Chan Medical School, Ferrari (Italy), IRCCS Istituto Auxologico Italiano, Istituti di Ricovero e Cura a Carattere Scientifico, University of Milan, Centro de Investigación Biomédica en Red, Centre for Biomedical Network Research on Rare Diseases, Research Institute Hospital 12 de Octubre, Humanitas University, Southampton General Hospital, University of Southampton, University of Sheffield, University of Oxford, Trinity College Dublin, Emory University, Imperial College London, Fondazione IRCCS Istituto Neurologico Carlo Besta, Hammersmith Hospital
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